5′-Guanylic acid (GMP) rivals 5′-inosinic acid (IMP) as the most widely used flavor enhancer. 5′-GMP is one of the substances responsible for the taste of mushrooms. On its own, 5′-GMP does not have much taste, but its effect is noticeable when used in combination with monosodium l-glutamate (MSG). 5′-GMP creates a synergistic flavor-enhancing effect in combination with 5′-IMP.
Several methods are known for producing 5′-GMP, including: (1) the extraction of RNA from yeast and the enzymatic digestion thereof, (2) microbial fermentation for the direct production thereof, (3) microbial fermentation for forming guanosine, followed by the chemical phosphorylation of guanosine, (4) microbial fermentation for forming guanosine, followed by microbial phosphorylation of guanosine, (5) microbial fermentation for the production of 5′-XMP, followed by the conversion of 5′-XMP to 5′-GMP using Corynebacterium spp, or (6) microbial fermentation for the production of 5′-XMP, followed by the conversion of 5′-XMP to 5′-GMP by E. coli. Of these methods, Method (1) has problems related to material supply and economy, and Method (2) suffers from the disadvantage of having a low production yield because cell membranes are impermeable to 5′-GMP. For these reasons, the other methods are typically applied in industry.
In vivo, for the conversion of 5′-XMP to 5′-GMP, as in the case of Methods (5) and (6), 5′-XMP aminase is responsible, which catalyses the following reactions (Pantel et al. (1975), J. Biol. Sci., 250(7), 2609-2613).

5′-XMP aminase is a member of the glutamine amidotransferase superfamily. Glutamine amidotransferases hydrolyze glutamine at the gamma-amide group to generate ammonia. The resulting free ammonia is assimilated into amino acids, nucleotides, sugars, coenzymes, and the like through polymerization reactions. Glutamine amidotransferases have many various target substances, but the method by which glutamine is hydrolyzed to form ammonia has been well conserved during evolution. Glutamine amidotransferases have been divided into two subfamilies: class I and class II. The class I enzymes includes anthranilate synthase, carbamoyl phosphate synthetase, CTP synthetase, formylglycinamidine synthetase, 5′-xanthylic acid aminase, imidazole glycerol phosphate synthase, aminodeoxychorismate synthase, and p-aminobenzoate synthase. All of these enzymes use, in addition to glutamine, external ammonia as an amine donor (Cell Mol. Life Sci. 54, 205-222, 1998). Unlike how ammonia, free from glutamine, is transferred to a substrate, the external ammonia is considered to directly transfer to transferase.
In the context of protein structure, 5′-XMP aminase can be separated into two well-defined domains: one having glutaminase activity responsible for catalytic hydrolysis of glutamine and the other domain having transferase activity (Nat. Str. Biol. 3(1), 74-86, 1996). The N-terminal domain with glutaminase activity is structurally similar to carbamyl phosphate synthetase, which has been well studied. The glutaminase activity is mainly achieved by a catalytic triad of cysteine, histidine and glutamate residues, which is similar to the catalytic mechanism of cysteine protease (Cell Mol. Life Sci. 54, 205-222, 1998). Particularly in E. coli, cysteine 86, histidine 181 and glutamic acid 183 form a catalytic triad. In the enzymatic mechanism of glutaminase, the catalytic cysteine residue forms a gamma-glutamyl thioester bond with glutamine, with the histidine serving as a base for the hydrolysis of glutamine into glutamic acid and ammonia (Fukuyama et al. Biochemistry 3, 1448-1492, 1964; von der Saal et al. Biochemistry 24, 5343-5350, 1985). Through a channel formed in the enzyme, this ammonia participates in the conversion of 5′-xanthylic acid to 5′-guanylic acid (Raushel et al. Biochemistry, 38(25), 7891-7899, 1999).
XMP aminase-catalysed conversion of 5′-XMP to 5′-GMP using ammonia shows the same mechanism as that of the reaction using L-glutamine, but is subtly different in properties. 5′-XMP aminase, although optimal at pH 8.3 for both substrates, exhibits two or more times as much catalytic activity for L-glutamine as for ammonia (Pantel et al. (1975), J. Biol. Sci., 250(7), 2609-2613). The difference increases as the reaction pH approaches neutral, which implies that 5′-XMP aminase does not employ a solution phase of ammonia (NH3), but takes advantage of L-glutamine in the conversion of 5′-XMP into 5′-GMP in vivo.
When treated with the cysteine-reactive sulfhydryl reagent Iodoacetamide or with the glutamine derivative chloroketone or acivicin, the activity of 5′-XMP aminase decreases with L-glutamine, but remains unchanged with ammonia, indicating that the cysteine residue at the active site of the glutaminase is essential for the glutamine-dependent activity of 5′-XMP aminase, but not for the ammonia-dependent activity of 5′-XMP aminase (Zalkin and Truitt, J. Biol. Sci. 252(15), 5431-5436, 1977; Massiere and Badet-Denisot, Cell Mol. Life. Sci. 54, 205-222, 1998).
In the case of anthranilate synthase, which belongs to the same class as 5′-XMP aminase, it is reported that the replacement of the conserved cysteine residue with glycine abolishes the glutamine-dependent anthranilate synthase activity but not the NH3-dependent activity of the enzyme (Paluh et al., J. Biol. Chem. 260, 1889-8601, 1985). Also, when the conserved cysteine residue of para-aminobenzoate synthase is replaced by serine, the production of the γ-glutamyl thioester adduct is attenuated, which leads to a decrease in the production of aminodeoxychorismate (Roux et al., Biochemistry, 32, 3763-3768, 1993). As for carbamoyl phosphate synthetase, its glutamine-dependent activity also disappears when the conserved cysteine residue is replaced with serine or glycine (Rubino et al., J. Biol. Chem., 261, 11320-11327, 1986).
Typically, since native enzymes have evolved to have activity suitable for cells, they often exhibit properties unsuitable for industrial applications due to their low activity. To overcome this problem, gene cloning of an enzyme of interest and the overexpression thereof have typically been studied in the art. In practice, an 5′-XMP aminase gene (guaA) was successfully isolated from wild-type Escherichia coli and cloned into an inducible expression plasmid which can be applied for the production of 5′-GMP from 5′-XMP (Biosci. Biotech. Biochem. 61(5), 840-845, 1997).
Another method of increasing protein expression of wild-type bacteria using drug resistance is described in Korean Pat. Laid-open Publication No. 2000-0040840. In this publication, a mutant strain having enhanced activity of 5′-XMP aminase, which is prepared by imparting decoyinine resistance to a wild-type Escherichia coli strain, is provided for increasing the expression of a gene of interest.
Inducible expression vectors for general use require expensive expression inducers such as IPTG, and are thus not suitable for industrial applications involving protein production on a large scale. A constitutive expression system arises as a solution to this problem. A great number of constitutive expression systems have been reported. In particular, a novel constitutive expression promoter was developed for Corynebacterium ammoniagenes known to be suitable for the fermentative production of nucleic acids (Korean Pat. Application No. 2004-107215). The constitutive expression systems are useful because they sustain the expression of an introduced protein for a cultivation period of host cells without the use of an expression inducer. However, when the overexpression of an introduced protein affects the growth of host cells, the cells stop growing, or a vector introduced into the cells is removed, resulting in low expression efficiency. The same results have been reported for 5′-XMP aminase (Biosci. Biotech. Biochem. 61(5), 840-845, 1997).
The growth halt or the vector removal in the constitutive expression system of 5′-XMP aminase is, in the opinion of the present inventors, attributable to the cytotoxicity of the constitutively overexpressed product. As a solution for circumventing this problem, the present inventors suppressed the glutaminase activity of 5′-XMP. As mentioned above, since 5′-XMP aminase utilizes L-glutamine to convert 5′-XMP into 5′-GMP within cells, an L-glutaminase activity-suppressed 5′-XMP aminase mutant has decreased activity in, and thus low toxicity to, cells. Furthermore, the 5′-XMP aminase mutant retains ammonia-dependent activity although it loses its glutamine-dependent activity, so that it is applicable for the industrial conversion of 5′-XMP into 5′-GMP. Through scrutiny into the biochemical mechanism of 5′-XMP aminase, the present inventors have developed a glutaminase-suppressed ammonia-specific 5′-XMP aminase and successfully realized its enhanced activity in culture fluid, leading to the present invention.